Ultrafiltration membranes and methods of making

ABSTRACT

The present invention is an integral multilayered composite membrane having at least one ultrafiltration layer made by cocasting or sequentially casting a plurality of polymer solutions onto a support to form a multilayered liquid sheet and immersing the sheet into a liquid coagulation bath to effect phase separation and form a multilayered composite membrane having at least one ultrafiltration layer.

CROSS-REFERENCED TO RELATED APPLICATIONS

The present application relates to U.S. Provisional Application No.60/686,363, filed on Jun. 1, 2005 and to U.S. Provisional ApplicationNo. 60/583,209, filed on Jun. 25, 2004 the entire contents of which areincorporated herewith.

This invention provides for multilayered composite membranes having atleast one ultrafiltration layer produced from at least two polymersolutions, and a novel method of manufacturing such membranes. Themembranes are particularly suited for use in dead-end ultrafiltration.

BACKGROUND OF INVENTION

Ultrafiltration and microporous membranes are used in pressure-drivenfiltration processes. Practitioners in the field of separation processesby membranes easily differentiate between microporous andultrafiltration membranes and generally distinguish between them basedon their application and aspects of their structure. Microporous andultrafiltration membranes are made, sold and used as separate anddistinct products. Despite some overlap in nomenclature, they areseparate entities, and treated as such in the commercial world.

Ultrafiltration membranes are primarily used to concentrate or diafiltersoluble macromolecules such as proteins, DNA, starches and natural orsynthetic polymers. In the majority of uses, ultrafiltration isaccomplished in the tangential flow filtration (TFF) mode, where thefeed liquid is passed across the membrane surface and those moleculessmaller than the pore size of the membrane pass through (filtrate) andthe rest (retentate) remains on the first side of the membrane. As fluidalso passes through there is a need to recycle or add to the retentateflow in order to maintain an efficient TFF operation. One advantage ofusing a TFF approach is that as the fluid constantly sweeps across theface of the membrane it tends to reduce fouling and polarization of thesolutes at and near the membrane surface leading to longer life of themembrane.

Microporous membranes are primarily used to remove particles, such assolids, bacteria, and gels, from a liquid or gas stream in dead-endfiltration mode. Dead-end filtration refers to filtration where theentire fluid stream being filtered goes through the filter with norecycle or retentate flow. Whatever material doesn't pass through thefilter is left on its upper surface.

Ultrafiltration membranes are generally skinned asymmetric membranes,made for the most part on a support which remains a permanent part ofthe membrane structure. The support can be a non-woven or woven fabric,or a preformed membrane.

Microporous membranes are produced in supported or unsupported form.Usually, the support has the membrane or a portion of the membraneformed in the support, rather than on the support, as in ultrafiltrationmembranes.

The early cellulosic, nylon and polyvinylidene fluoride microporousmembranes were symmetric and for the most part, unskinned. Presently,some asymmetric microporous membranes are produced, and some of theseare skinned.

While it would seem that the two types of membrane could bedifferentiated by pore size, this is not the case, as will be discussedbelow. The reasons for this are that they are used in differentapplications, requiring different characterization methods. None of themethods usually used give an absolute pore size measure, and differentmethods cannot be directly compared.

Despite the similarities between microporous membranes andultrafiltration membranes, the history of their development is quitedifferent. It is therefore not surprising that there is more than oneaccepted demarcation between them.

Microporous membranes were commercially developed from the work ofZsigmondy by Sartorius Werke (Germany) in 1929. These were what are nowcall “air cast” membranes made by evaporating a thin layer of a polymersolution in a humid atmosphere. These membranes were and still aresymmetric and generally unskinned. Since they were used to remove orhold bacteria, they were rated by the bacteria size that would beretained. This method resulted in pore size ratings in microns.

A common method used to rate microporous membranes is the bubble pointtest. In this method, the microporous membrane is placed in a holder andsaturated with a test liquid. Gas pressure is applied to one side of themembrane and the pressure is increased at a fixed rate. The appearanceof the first stream of bubbles from the downstream side is a measure ofthe largest pore. At a higher pressure where the liquid is forced out ofthe majority of the pores, the foam all over point (FAOP) is reached.These are described in ASTM F316-70 and ANS/ASTM F316-70 (Reapproved1976).

Ultrafiltration membranes (UF) are a spin-off of the reverse osmosismembrane development research of Leob and Sourirajan. Alan Michaelsfixed 1965 as the time when the first rudimentary UF membranes anddevices first appeared on the market. UF membranes are made by immersioncasting methods and are skinned and asymmetric. The initial commercialapplications were related to protein concentration and the membraneswere rated by the molecular weight of the protein that they wouldretain, i.e., the molecular weight cutoff rating of the membrane (MWCO).

While membrane ratings based on testing with proteins is still done, acommon method uses non-protein macromolecules having a narrow molecularweight distribution, such as polysaccharides (Dextrans) or polyethyleneglycols. See for example, A rejection profile test for ultrafiltrationmembranes and devices, BIOTECHNOLOGY 9 (1991) 941-943.

As membrane applications were developed in the 1960's and 1970's, UFmembranes expanded to larger pore sizes and microporous membranes (MF)to smaller pores sizes. As this occurred, practitioners began todifferentiate between the two types of membranes. It is interesting froma historical perspective that the earliest literature referred only toultrafiltration. Both Kesting Synthetic Polymer Membranes A StructuralPerspective, Robert E. Kesting, John Wiley & Sons 1985 and Lonsdale “TheGrowth of Membrane Technology”. K. Lonsdale, J. Membrane Sci. 10 (1982)81 cite to Ferry's major review of 1936 in which ultrafiltration refersto both ultrafiltration and microfiltration membranes. Kesting states“The term ultrafiltration has changed its meaning over the years.” Infact, even in a 1982 review Pusch Synthetic Membranes—Preparation,Structure, and Application, W. Pusch and A. Walch Angew. Chem. Int. Ed.Engl. 21 (1982) 660 uses ultrafiltration to denote sieving membranes offrom 0.005μ to 1μ. Kesting, in table 2.9 (pg 45) has UF as 10-1000Angstroms, 0.01-0.1 microns, and MF as 1000-100.000 Angstroms, 0.1-10microns.

A 1969 chart from Dorr-Oliver has microporous pore size ranging from0.03μ to over 10μ, and UF ranging from 0.002μ to 10μ. A recent handbookchapter. Handbook of Separation techniques for Chemical Engineers—ThirdEdition, Section 2.1 Membrane Filtration, M. C. Porter, McGraw-Hill 1996claims this “reflects confusion in the literature among MF, UF and RO.”In 1975 Porter Selecting the Right Membrane, M. C. Porter, Chem. Eng.Sci. 71 (1975) 55 proposed that UF cover the range from 0.001 to 0.02microns, and MF from 0.02 to 10 microns. Lonsdale referred to this inReference 2 and Porter uses this definition again in reference 4.

Cheryan Ultrafiltration Handbook, M. Cheryan, Technomic Publishing Co.Chapter 26—Introduction and Definitions (Ultrafiltration) S. S. Kulkarniet al Chapter 31—Definitions (Microfiltration) R. H. Davis 1986 has bothPorter's ranges for UF and MF (uncited) and a chart that appears to befrom the Dorr-Oliver chart. In Membrane Handbook, Davis, Van Nostrandand Reinhold DATE Davis gives MF as 0.02-10 microns, and Kulkarni et aldescribe UF as 10 to 1000 Angstroms, 0.001-0.1 microns. Another exampleof pore size ranges is from the Encyclopedia of Polymer Science andEngineering, Volume 9 pg 512, John Wiley and Sons 1987 which has UF asfrom 0.01 to 0.1 microns and MF as from 0.1 to 10 microns. ZemanMicrofiltration and Ultrafiltration, L. Zeman and A. Zydney, MarcelDekker, Inc 1996, p 13 has a chart in which UF ranges from 0.001 to 0.1micron and MF from about 0.02 to 10 microns.

With respect to the present invention, we will define ultrafiltrationmembranes as compared to microporous membranes based on the definitionsof the International Union of Pure and Applied Chemistry (IUPAC),“Terminology for membranes and membrane processes” published in PureAppl. Chem., 1996, 68, 1479.

“72. microfiltration: pressure-driven membrane-based separation processin which particles and dissolved macromolecules larger than 0.1 μm arerejected”

“75. ultrafiltration: pressure-driven membrane-based separation processin which particles and dissolved macromolecules smaller than 0.1 μm andlarger than about 2 nm are rejected.”

The definition for ultrafiltration membranes will be based on what theydo, and how they do it. Ultrafiltration membranes are capable ofconcentrating or diafiltering soluble macromolecules that have a size insolution of less than about 0.1 micron and operating continuously in atangential flow mode for extended periods of time, usually more than 4hours and for up to 24 hours. Microporous membranes are capable ofremoving particles larger than 0.1 micron and being used in dead-endfiltration applications. Microporous membranes generally allow solublemacromolecules to pass through the membrane.

Ultrafiltration membrane production methods by immersion casting arewell known. A concise discussion is given in Microfiltration andUltrafiltration: Principles and Applications Marcel Dekker (1996); L. J.Zeman and A. J. Zydney eds. These preparations are generally describedto consist of the following steps: a) preparation of a specific and wellcontrolled preparation of a polymer solution, b) casting the polymersolution in the form of a thin film onto a substrate, c) coagulating theresulting film of the polymer solution in a nonsolvent and d) optionallydrying the ultrafiltration membrane.

The common form of ultrafiltration membranes is the asymmetric membrane,where the pore size of the membrane varies as a function of locationwithin the thickness of the membrane. The most common asymmetricmembrane has a gradient structure, in which pore size increases from onesurface to the other. Asymmetric membranes are more prone to damage,since their retention characteristic is concentrated in a thin surfaceregion or skin. A membrane skin is a thin dense surface penetrated bysurface pores. It has been found, however, that increased productivityresults from having the feed stream to be filtered contacting the largerpore surface, which acts to prefilter the stream and reduce membraneplugging.

Practitioners in the art of making ultrafiltration membranes,particularly asymmetric membranes, have found that membranes whichcontain large (relative to membrane pore size) hollow cavernousstructures have inferior properties compared to membranes made withoutsuch hollow structures. These hollow structures are sometimes called“macrovoids”, although other terms are used in the art. Practitionersstriving for membranes of very high retention efficiency prefer to makemembranes without such hollow structures.

Perhaps the most direct variation of the single layer structure is amultilayered unbonded laminate. While laminates can be made from layersof the same or different membranes, they have drawbacks. Each layer hasto be made in a separate manufacturing process, increasing cost andreducing manufacturing efficiency. It is difficult to manufacture andhandle very thin membranes, less than say 20 microns, because theydeform and wrinkle easily. This adds to the inefficiency of producing afinal product with thin layers. Unbonded laminates can also come apartduring fabrication into a final filter device, such as a pleated filter,which will cause flow and concentration non-uniformities. Other methodsof forming multilayered porous membrane structures are known. U.S. Pat.No. 4,824,568 describes a composite ultrafiltration membrane made bycasting a thin ultrafiltration membrane onto a preformed microporousmembrane. U.S. Pat. No. 5,228,994 describes a method for coating amicroporous substrate with a second microporous layer thereby forming atwo layer composite microporous membrane. These processes require twoseparate membrane forming steps, forming one on top of the otherpreformed membrane and are restricted by the viscosities of the polymersolutions that can be used in the process to prevent excessivepenetration of casting solution into the pores of the preformedsubstrate.

In U.S. Pat. No. 5,620,790, a method of making a microporous membrane isdescribed wherein the membrane is made by pouring out a first layer on asupport of polymeric material onto a substrate and subsequently pouringout one or more further layers of a solution of polymeric material ontothe first layer prior to the occurrence of turbidity in eachsuccessively immediate preceding layer, the viscosity of eachimmediately successive layer of a solution of polymeric material havingbeen the same or less than that of the preceding layer. US PatentApplication 20030217965, directed to microporous membranes, provides fora method of producing an integral multilayered porous membrane bysimultaneously co-casting a plurality of polymer solutions onto asupport to form a multilayered liquid sheet and immersing the sheet intoa liquid coagulation bath to effect phase separation and form a porousmembrane. U.S. Pat. No. 6,706,184 discloses a process for forming acontinuous, unsupported, multizone phase inversion microporous membranehaving at least two zones comprised of the acts of: operativelypositioning at least one dope applying apparatus, having at least twopolymer dope feed slots, relative to a continuous moving coatingsurface; applying polymer dopes from each of the dope feed slots ontothe continuously moving coating surface so as to create a multiple layerpolymer dope coating on the coating surface; subjecting the multipledope zone layer to contact with a phase inversion producing environmentso as to form a wet multizone phase inversion microporous membrane; andthen washing and drying the membrane. In these structures, each layer orzone is a microporous membrane. US Patent Application 20040023017describes a multilayer microporous membrane containing a thermoplasticresin, comprising a coarse structure layer with a higher open pore ratioand a fine structure layer with a lower open pore ratio, wherein saidcoarse structure layer is present at least in one membrane surfacehaving a thickness of not less than 5.0μ, a thickness of said finestructure layer is not less than 50% of the whole membrane thickness,and said coarse structure layer and said fine structure layer are formedin one-piece. The fine structure is not skinned. This structure isformed from a single solution.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates a co-casting coating head

FIGS. 2 a and 2 b illustrate the position of the layers for two layermembranes

FIG. 3 shows results from filtration of fluorescent beads

FIGS. 4 a-4 d show scanning electron micrograph images of the membranesof Examples 1 and 2.

FIG. 5 shows scanning electron micrograph images of the membranes ofExample 3.

SUMMARY OF THE INVENTION

This invention comprises an integral multilayer flat sheet membrane madefrom more than one polymer solution, wherein at least one layer is anultrafiltration membrane. The method of making the membrane is includedin the present invention

In an embodiment, the invention comprises a skinned asymmetricultrafiltration membrane layer joined to a microporous membrane layer,where the junction has a gradient of pore sizes, transitioning from thepore size of the microporous layer in the vicinity of the junction tothe pore size of the ultrafiltration layer in the vicinity of thejunction.

In an embodiment, the invention comprises a microporous membrane layerjoined to the tight pore side of an ultrafiltration layer, where thejunction has a gradient of pore sizes, transitioning from the pore sizeof the microporous layer in the vicinity of the junction to the poresize of the ultrafiltration layer in the vicinity of the junction.

In an embodiment, the invention comprises a skinned asymmetricultrafiltration membrane layer joined to a second asymmetricultrafiltration membrane layer, the second ultrafiltration membranehaving an average retentive pore larger than that of the skinnedasymmetric first layer, where the junction has a gradient of pore sizes,transitioning from the pore size of the second ultrafiltration layer inthe vicinity of the junction to the pore size of the firstultrafiltration layer in the vicinity of the junction.

In an embodiment, the invention comprises a process for forming anintegral multilayered composite ultrafiltration membrane compromisingthe steps of operatingly positioning a polymer solution applyingapparatus having at least two dispensing outlets relative to a movingcarrier surface, and; supplying each dispensing outlet with a differentpolymer solution, and; applying said solutions onto said moving carriersurface so as to create a multiple layer coating on said carrier, andwherein; said multiple layers are dispensed with essentially no timeinterval between successive layers being applied, and; subjecting saidmultiple liquid layers to a phase separation process so as to form a wetmultilayer ultrafiltration membrane.

The invention further embodies the use of the membranes of the presentinvention in a process to remove viral particles from a manufacturedprotein-containing solution, made in the course of producing biotechderived pharmaceuticals, wherein the membranes is each capable ofsubstantially preventing the passage therethrough of the virus particlesand substantially permitting the passage therethrough of the protein,

DETAILED DESCRIPTION OF THE INVENTION

The present invention is an integral multilayered composite membranehaving at least one ultrafiltration layer made by cocasting a pluralityof polymer solutions onto a support to form a multilayered liquid sheetand immersing the sheet into a liquid coagulation bath to effect phaseseparation and form a porous ultrafiltration membrane. After formation,the porous membrane is washed free of solvent and other solublematerials. It can then be further extracted to reduce fugitive materialsto a low level and then optionally be dried.

In order to reduce the present invention to practice, the inventors hadto overcome the fundamental problem of making an integral multilayeredstructure from markedly dissimilar membrane-forming solutions. Theinventors faced practical difficulties associated with the differencesin structure and formation of the dissimilar membrane layers. While theprior art describes methods of making multilayered microporousmembranes, in that art the membrane formulation solutions and mechanismsof membrane formation and membrane structure of each layer are quitesimilar.

In the present invention, pore size differences between theultrafiltration (UF) layer and the microporous (MF) layer can differ byan order of magnitude. Also, the rate of formation of UF and MFmembranes is different, with UF forming significantly faster in thecoagulation bath. Potential and real problems that can arise with thefabrication of membranes of the present invention include:

If the last solution coated is a UF-forming solution, it may form toofast and too densely to permit the coagulation liquid to penetrating tothe microporous forming layer at a rate necessary to form a satisfactorymembrane. If the coagulant diffuses through the top layer too slowly,formation of the underlying microporous layer will be hindered, and poresize will be uncontrollable. As well, it may even occur that theunderlying layer may not solidify before the coated sheet exits from thecoagulant bath.

If the MF-forming solution is coated over the UF forming layer, theformation of the microporous layer will greatly affect the formation ofthe UF layer. It will prevent the formation of the skin surface, andchange the pore size that would have resulted if the UF layer were castseparately.

Other problems can result from the disparity between viscosity rangesfor the two types of membranes. UF membrane forming solutions areusually of much higher viscosity than those for MF membranes. Coatingmultilayers with viscosity discrepancies only exacerbates the problemsof making porous membranes

The inventors have found that within certain limited ranges on keyvariables, integral multilayer cocast composite ultrafiltrationmembranes with practical properties can be made.

For simplicity, the process of making a multilayer cocast compositeultrafiltration membrane will be described for a two layer example.Although three or more layers may also be made by the same process. Thepreferred process comprises the steps of making two polymer solutions,one for each layer. Solutions for making porous membranes by immersioncasting usually comprise a polymer, solvent and additives to modify andcontrol the final pore size and porous nature (i.e., percent porosity,pore size distribution, etc.) of the membrane. Other additives aresometimes used to modify physical properties such as hydrophilicity,elongation, modulus, etc.

Preferred polymers include but are not limited to polyvinylidenefluoride (PVDF), nylons such as Nylon 66, polyamides, polyimides,polyetherimides, polyethersulfones, polysulfones, polyarylsulfones,polyphenylsulphones, polyvinylchlorides (PVC), polycarbonates,cellulose, regenerated cellulose, cellulose esters such as celluloseacetate or cellulose nitrate, polystyrenes, acrylic polymers,methacrylic polymers, copolymers of acrylic or methacrylic polymers, orblends of any of the above and the like.

Solvents used include but are not limited to such examples as dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, tetramethylurea,acetone, dimethylsulfoxide and triethyl phosphate.

Examples of the many porogens have been used in the art, include but arenot limited to compounds such as formamide, various alcohols andpolyhydric compounds, water, various polyethylene glycols, polyvinylpyrrolidone and various salts, such as calcium chloride and lithiumchloride.

Examples of other additives include surfactants to improve wettability,and polymers compatible with the primary membrane polymer used to modifymechanical properties of the final membrane.

After the solutions are made, they are applied to a moving carrier. Foran unsupported membrane, which does not have a web attached to the finalmembrane, the carrier is usually a plastic film, such as polyethyleneterephtalate, or a polyethylene coated paper, or similar smoothcontinuous web that can be easily removed from the formed membrane.

Application can be done by any standard method. The object is to coat afirst solution onto the carrier and a second solution upon the firstsolution. A highly preferred method is co-casting, in which the twolayers are coated with essentially no time between coatings. This can bedone with a double knife over roll apparatus a pressurized dual slotcoating head or any other pre or postmetering coating device as is knownin the industry. Co-casting means that the individual layers are castessentially simultaneously with each other with substantially no timeinterval between one cast layer and the next cast layer. This method isdescribed in detail in US published Patent Application 20030217965.Co-casting is an important aspect of the invention because it allows forformation of controlled pore size regions at the junctions of layers. Inthe prior art, a well-defined demarcation line is formed between thesequentially cast layers. A drastic change in pore size going from amore open to a more tight structure can lead to undesirable fastaccumulation of retained solute at the interface and consequently adrastic flux decline. Possibly due to partial mixing of adjacent co-castlacquers or due to high shear forces at the interface between twoadjacent co-cast lacquers, a sharp interface can be replaced by a moresubtle change in pore size between two adjacent layers with a cocastprocess. Such an interfacial zone is beneficial for the retentivebehavior of the overall structure of the multilayered membrane and istherefore preferable in some applications. However even with the cocasttechnique one can, if desired, form a sharp or well-defined demarcationline between layers with the proper selection of materials andapplication methodologies.

The membranes of the present invention are preferably produced using apremetered coating process. Premetered coatings are those in which theexact amount of coating solution to be deposited is fed to the coatinghead. The height of the layers are set by the deposition rather than bysome post application means such as a doctor blade which sets thethickness of the structure after metering of the layers (commonlyreferred to as “post metering process”). The premetered term is appliedto die coating, slide and curtain coating among other methods of formingthe structure. The present invention preferably uses a double knife boxor double slot die. Post metered applications can also be used ifdesired.

FIG. 1 illustrates a multiple layer forming apparatus 10 for castingmultilayered membranes. As shown, the apparatus is designed to produce atwo-layered liquid film and has two chambers 50 and 60 containing thesolutions 14 and 16, one for each layer, to be cast. If desired,additional chambers may be added to form additional co-cast layers. Theapparatus comprises a front wall 20 and a back wall 40 with a separatingwall 30 between the front and back walls. The separating wall definesthe volumes of the two chambers. Two side walls, not shown, complete theapparatus. In operation, the apparatus is fastened onto a typicalmembrane casting machine, and a support web 18 is moved or passed underthe stationary apparatus and the two solutions are dispensed throughgaps or outlets 80 and 90. The thickness of the two layers is controlledby the distance (gap) set between the moving web and the outlet,illustrated by gap settings 80 and 90. The final liquid layer thicknessis a function of gap distance, solution viscosities, and web speed. Theback wall of the apparatus usually is held a small distance above thesupport to prevent wrinkling or marring the support. Back wall gap,support speed and solution viscosity are adjusted in practice to preventsolution from leaking out through the back wall gap. The apparatus canbe fitted with heating or cooling means for each chamber separately, orfor the apparatus as a whole, if necessitated by the solutioncharacteristics, or to further control final membrane properties.

A slot die consists of an enclosed reservoir with an exit slot having asmaller cross-section. An extruder or positive displacement pump, or insome cases a pressurized vessel feeds the coating into the reservoir ata uniform rate, and all of the fluid that goes into the die is forcedout from a reservoir through a slot by pressure, and transferred to amoving carrier web. The slot is positioned perpendicular to the movingcarrier web. Multiple layer coatings require a die with individualreservoirs, and associated feed method, and exit slots for each layer.

After the layers are coated onto the moving carrier, the carrier withthe liquid sheet is immersed into a liquid that is a nonsolvent for thepolymer, and miscible with the solvent and porogens. This will causephase separation and the formation of a porous membrane.

The formed composite membrane is then usually separated from the carrierand washed to remove residual solvent and other material. The membranecan then be dried. Ultrafiltration membranes are usually dried with ahumectant, such as glycerine, by first immersing the washed membrane inan aqueous glycerine solution, of from 5% to 25% concentration byweight, and removing excess liquid, before proceeding through the dryingstep. Drying is done in a manner to remove the majority of the water andto leave sufficient glycerine to prevent pore collapse.

In the coagulation of a multilayered liquid sheet, coagulation occursfrom the liquid film surface that first contacts the coagulation bathand then through the subsequent layers of the multilayered liquid sheet.Each layer dilutes and changes the coagulant as the coagulant diffusesthrough the layers. Such changes to the nature of the coagulant affectthe membrane formation of each layer and of the final multilayermembrane. Layer thickness, composition and location of each layerrelative to the other layers will affect membrane structure andproperties. Each layer forms differently than it would if it were to bemade from a single layer solution or from laminates of single layers.

In another embodiment, the two or more layers are sequentially castsuccessively on to the prior cast layer with some time between each castso that some phase separation may occur in the earlier cast layer. Allother steps of the process are the same as for those described with thecocast embodiment. This embodiment of sequential casting allows one toform UF containing structures similar to embodiments using onlycocasting methods.

Reference is made to FIGS. 2 a and 2 b as an aid in the description ofthese multilayered membranes. It is common convention of those skilledin the art to denote as the “top surface” of an asymmetric membrane thefacial surface having the smallest pore size. We will use thisconvention as a basis. For the case of multilayered membranes having anskinned asymmetric ultrafiltration membrane layer with no other layerscontacting the skinned surface, the skinned asymmetric ultrafiltrationmembrane layer will be the first layer, or top layer, and subsequentlayers numbered two, three, etc. This is illustrated in FIG. 2 a. To beconsistent, for the case where a microporous membrane is the top layerover an asymmetric ultrafiltration layer, the microporous layer will bedenoted as the first layer, the ultrafiltration layer the second layerand so on. This is illustrated in FIG. 2 b. Another manner ofequivalently describing the nomenclature is to denote that the firstside will be the top layer, i.e., the last solution coated onto thecarrier, of the multilayered liquid sheet that has been cast.

The multilayered membrane of the present invention is not the same as anadditive series of equivalently made single layer membranes. Due to theintegral joining of the layers, there is a region where the pore sizetransitions from one layer to the next. To describe the structures, wewill use the following device, with a two layer membrane as an example.A single layer membrane consists of a first side, a second side, and aporous structure between. Similarly, a laminate of two membranes wouldconsist of a first layer with a first side, a second side, and a porousstructure between, and a second layer with a first side, a second side,and a porous structure between. For a two layer membrane of the presentinvention, the first layer has a first side, and a second equivalentside. The second equivalent side would be a second side if this layerwas a single layer membrane, but here it is part of the integral joiningof the two layers. Likewise, the second layer has an equivalent firstside and a second side and a porous structure between. The twoequivalent sides are conjoined to form the conjoined thickness, that is,the transition zone between the two layers.

Asymmetric ultrafiltration membranes are sometimes used in dead-endfiltration with the open or large pore surface at the upstream or highpressure side. An important application for such use is in removal ofviral particles from process solutions in the manufacture of biotechtherapeutic drugs. This is described in U.S. patent application Ser. No.10/145,939.

The advantage of dead-end filtration lies in its simplicity. Thepressurized feed stream is contacted with one side of the membrane andthe fluid passes through while the material to be removed is retained bythe membrane. In comparison, in tangential flow filtration (TFF), thepressurized feed stream is directed tangentially across the membraneface, and a portion of the feed stream passes through the membrane,while the remainder, the retentate, is usually recycled with addedmake-up feed, or returned to the feed tank. TFF requires extra pumpingequipment, and more controllers to maintain the proper ratios of flowsand pressure. However, dead-end filtration with ultrafiltrationmembranes has not been commonly used because the membranes tended tolose permeation properties too quickly to be useful.

The inventors have found that the multilayer ultrafiltration membranesof the present invention have greatly improved properties over prior artultrafiltration membranes. The apparent reason for this improvement liesin the structure of the membranes, although no limitation should be puton the scope of the invention by the following discussion. It appears tothe inventors that the pore size transition in the conjoining regionplays a key role in the improved properties.

This is illustrated in FIG. 3 in which an example of a membrane of thepresent invention, a skinned asymmetric ultrafiltration membraneconjoined to a microporous layer, is compared to a two layerpolyvinylidene fluoride (PVDF) ultrafiltration membrane made by castingan ultrafiltration layer on a pre-formed microporous membrane,(Viresolve® membrane available from Millipore Corporation of Billerica,Mass.).

FIG. 3 shows crossectional views of the membranes after having been usedto filter fluorescent polystyrene beads from the open pore side in adead-end mode. Three tests were done with bead sizes of 31 nm, 60 nm,and 170 nm.

For testing done with the 31 nm particles with the membrane of thepresent invention, the filtered particles are distributed throughout thethickness of the ultrafiltration layer. However, for the two layer PVDF,the particles are concentrated just under the small pore surface of themembranes. Since this surface provides the limiting pore size for flow,the concentrated particle layer will be more likely to plug these poresand will have a more deleterious effect on permeation.

For testing with the 60 nm particles, the particles are held away fromthe ultrafiltration layer and are diffusely distributed in theconjoining region. For the two layer PVDF, the particles form aconcentrated layer near or at the junction of the microporous substrateand the ultrafiltration layer for the PVDF membrane.

Similar results are seen for the 170 nm particle testing. The membraneof the present invention retains the particles in a diffuse layer awayfrom the skin. The two layer PVDF again traps the particles in a denselayer at the junction of the two layers.

In all these cases, the membrane of the present invention trapsparticles in a diffuse manner, which spread out the effects of poreplugging and increase filter flow and lifetime.

In an embodiment of the present invention, we use the teachings of U.S.Pat. No. 5,444,097 ('097) in a novel manner. The '097 patent teaches theuse a polymeric solution exhibiting a lower critical solutiontemperature (LCST) to make microporous membranes. Heating an LCSTsolution above the LCST causes phase separation. This step isincorporated in the process of the present invention after themultilayered liquid film is formed to further vary and control thestructures of the resulting membrane layers. One or several of thesolutions of the present invention would be a LCST solution. It has beenfound that the temperature to which the solution is raised above theLCST, and the time the solution is held above LCST, controls the finalpore size of the membrane layer. Furthermore, if there is a temperaturegradient in a liquid layer, then there will be a corresponding pore sizegradient.

In the present invention, the use of LCST solutions is used to produce avariety of structures.

For an bilayer composite ultrafiltration membrane made using LCSTsolutions having a first layer of a skinned asymmetric layer on a secondmicroporous layer, a preferred solution for the first layer will have apolymer content of from about 15% to about 30% polymer solids, with amore preferred range of from about 20% to about 25% polymer solids. Allpercentages related to solutions are by % weight of the solution. Forthe microporous layer, the polymer solution will have a polymer contentof from about 10% to about 20% polymer solids, with a more preferredrange of from about 15% to about 18% polymer solids by weight of thesolution. The LCST of the first layer solution is preferably from about700 to about 150° C. For the second layer, the LCST range is preferablyfrom about 40° to about 60° C. The thickness of the first layer is fromabout 2 microns to about 100 microns, preferably 2 microns to about 50microns, with a more preferred range from about 2 microns to about 25microns. The microporous second layer has a thickness range of fromabout 50 microns to about 200 microns, with a preferred thickness offrom about 80 microns to about 150 microns, with a more preferred rangeof from about 100 microns to about 125 microns. It is preferable thatthe total thickness of the cocast composite membrane be in the range offrom about 52 microns to about 300 microns, preferably from about 75microns to about 200 microns, with a more preferred range of 90 micronsto about 120 microns. If the pore size is determined by the temperatureto which the LCST solution is raised above LCST, and the time maintainedabove LCST, the practitioner will determine by routine trial and errorthe proper conditions for operating their particular process equipment.Heating the solution can be done by several methods. The support coatedwith the polymer solution layers can be conveyed over a heated surface,such as a flat plate, a block, or a rod. A preferable method is to use arotating heated drum. Heating can also be done by non-contact methodssuch as for example, infrared heating or microwave energy. If a heateddrum is used to raise temperature of the coated web, the thickness andthermal insulating properties of the carrier web, and thickness of thepolymer solution will be germane to obtaining a desired pore size. Thetemperature of the drum and the speed of the process will then bedetermined and controlled to produce the desired membrane. Thetemperature of the heated surface is determined by the equipment and themanufacturing process conditions as described above.

For the case of a microporous first layer and a second ultrafiltrationlayer, a preferred solution for the first layer will have a polymercontent of from about 10% to about 20% polymer solids, with a morepreferred range of from about 12% to about 16% polymer solids. Allpercentages related to solutions are by % weight of the solution. Forthe second ultrafiltration layer, the polymer solution will have apolymer content of from about 15% to about 30% polymer solids, with amore preferred range of from about 20% to about 25% polymer solids. TheLCST of the first layer solution is preferably from about 40° to about60° C. For the second layer, the LCST range is preferably from about 70°to about 120° C. The thickness of the first layer is from about 2microns to about 50 microns, with a more preferred range from about 5microns to about 25 microns. The ultrafiltration membrane second layerhas a preferred thickness of from about 80 microns to about 150 microns,with a more preferred range of from about 100 microns to about 125microns. It is preferable that the total thickness be in the range offrom about 90 microns to about 120 microns. Similar to the above case,the practitioner will determine by routine trial and error the properconditions for operating their equipment.

If it is desired to make two layers from ultrafiltration formingsolutions, the parameters above will serve as guides to the individuallayer compositions and process parameters.

In an embodiment, the first layer is formed from a solution and underconditions that would give a skinned asymmetric ultrafiltration membraneif cast as one layer. The second layer would give a microporous membraneif cast as one layer. The resulting structure is a skinned asymmetricultrafiltration membrane on a microporous layer, with an integraltransition zone between them. In a preferred method, both solutions fromwhich the layers are cast have a LCST, with the ultrafiltration layerhaving a higher LCST. The cast multilayered liquid sheet is heated to apreplanned temperature above the LCST of the second (microporous) layerbut below the LCST of the first (ultrafiltration) layer before immersioninto the precipitation bath. This has been found to result in anultrafiltration layer over a microporous layer with a transition zonebetween.

A preferred version of this embodiment can be done where theultrafiltration layer does not have a LCST, or does not have ameasurable LCST, while the microporous layer solution has a LCST, andthe same general structure will result.

It is also possible to use two solutions, neither of which have a LCST,but which individually would make the combination of ultrafiltration andmicroporous layers required.

In an embodiment illustrated by Example 3, the membrane is formed from atop layer ultrafiltration membrane made from a solution with an LCSThigher than the drum temperature used to heat the formed solution layersbefore immersion, and a bottom layer made from a solution having an LCSTlower than the drum temperature. In this example, the LCST of the UFlayer solution is assumed to be greater than 150° C. because it couldnot be measured due to limitations of the test equipment. Surprisingly,as the drum temperature approximately equals the LCST of the microporouslayer solution, the gradient between the two layers becomes lessobservable. However, the composite membrane so-formed out-performs a twolayered membrane made by casting an ultrafiltration layer on a preformedmicroporous membrane (Viresolve, Millipore Corporation). Without beinglimited by the following, it is the inventors present theory that theViresolve membrane-making process results in interpenetration of the toplayer into the bottom layer, which gives the type of results discussedin relation to FIG. 3. However, the membrane of Example 3, because it isformed in a single step, does not have the same type of “bottleneck” atthe interface of the two layers. In fact, it has a gradient, which,albeit sharp, still functions as described herein as a membrane of thepresent invention.

In an embodiment, the first layer is a microporous layer preferablythin, that is, between 5 to 30 microns thick, and the second layer ismade from a solution and under conditions that would produce anultrafiltration layer. In a highly preferred embodiment, the microporousand ultrafiltration layers are produced from solutions having a LCST,with the LCST of the ultrafiltration layer solution being higher. Whenheated above the LCST of the microporous layer solution, but below thatof the ultrafiltration layer solution, the microporous layer will phaseseparate. Subsequent immersion will fix the microporous structure andcause phase separation of the ultrafiltration solution to form themultilayered membrane. A preferred version of this embodiment can bedone where the ultrafiltration layer does not have a LCST, while themicroporous layer solution has a LCST, and the same general structurewill result. It is also possible to use two solutions, neither of whichhave a LCST, but which individually would make the combination ofultrafiltration and microporous layers required.

In an embodiment, the multilayered ultrafiltration membrane is made oftwo layers of ultrafiltration membrane-making solutions that would, ifcast as single layers, produce skinned asymmetric ultrafiltrationmembranes.

In a similar manner, solutions with an upper critical solutiontemperature (UCST) which phase separate when cooled below the UCST canbe used to make the inventive membranes, are formed into a multilayeredliquid film in a heated state and cooled to obtain phase separation. Inboth the LCST and UCST embodiments, further phase separation can beprovided by immersion into a coagulant, as described previously.

Control of the transition zone or region is important for the presentinvention. In order to get a useful transition zone, the inventors havefound that it is desirable to control the thickness of each layer, inparticular the first layer, as well as the relative viscosities of thetwo solutions, so that the viscosity difference is not too great, andthe relative time of formation, that is, solidification, of the layers.The variables above will serve as a guideline for other practitioners,but it must be appreciated that for each set of solutions and theparticular equipment used, there may be differences from those statedwithin the present description.

The present invention provides a high-resolution membrane-based methodfor removing a virus from a manufactured protein-containing solution,the method being particularly characterized by its capacity to beperformed quickly (i.e., as measure by flux) and efficiently (i.e., asmeasured by log reduction value, LRV).

Conduct of the methodology involves flowing a manufacturedprotein-containing solution through a filtration device containing thecomposite ultrafiltration membranes of the present invention underconditions sufficient to effect passage of said protein through saidcomposite membranes, and whereby any specifically-targeted viruscontaminating said protein-containing solution, is substantiallyprevented from passing through said asymmetric membranes, is therebysubstantially removed from the solution.

A “manufactured protein-containing solution” as used herein is a term ofspecific definition. In contrast to a solution havingnaturally-occurring protein content (e.g., water havingnaturally-occurring microbial content), the protein content in a“manufactured” solution will be enriched, as a result of humanintervention and possible conduct of other solution refinementprocesses, such that the predominant solute in said solution is saidprotein.

In respect of the composite membranes, several criteria need to bepresent to perform the inventive methodology. First, each must besubstantially hydrophilic. Secondly, the composite membranes must becapable of substantially preventing the passage therethrough of thetargeted virus, whilst substantially permitting the passage therethroughof the bio-manufactured protein

Aside from, but relevant to, the virus removal methodology, the presentinvention also provides a filtration capsule comprising a pleated tubeformed of one, two or three interfacially-contiguous compositeultrafiltration membranes. Although perhaps having applicabilityelsewhere, this product configuration has been found quite effective inthe conduct of the inventive virus removal methodology, in respect ofits durability, reliability, cost, and ease of use and replacement.

In light of the above, it is an objective of the present invention toprovide a methodology for removing at a high resolution a virus from amanufactured protein-containing solution, and particularly, one capableof being performed effectively at a log reduction value of greater than6 for a comparatively large virus (e.g., murine leukemia virus) or froma comparatively smaller virus (e.g., parvo virus).

It is another objective of the present invention to provide a filtrationcapsule useful for conducting said virus removal methodology.

It is another object of the present invention to provide a device forremoving a virus from a solution, the device comprising a housingsuitable for containing a filtration material and further characterizedby an inlet for receiving fluid to be filtered and an outlet forremoving filtrate, the filtration material comprising one, two or threecomposite void-free membranes, the upstream layer oriented such that its“tightest” side faces downstream.

In general, it has been found that by incorporation of multipleasymmetric ultrafiltration membranes, arranged in a pleatedconfiguration with the membranes in “tight side down stream”orientation, the resulting filter capsule will have good viral retentioncapabilities, yet maintain good flux. Although these may not be as highwithout using all the teaching underlying the inventive methodology,such high degree of accomplishment (particularly with respect to viralretention) is not always required in all circumstances. For example, forcertain non-pharmaceutical purification applications, log viralreduction values need not approach a value greater than 2.

As to its preferred structure, the filtration capsule comprises atubular housing and a pleated filtration tube substantially co-axiallyenclosed within said housing. The tubular housing of the filtrationcapsule is constructed to contain and channel a fluid process streamconducted there through—and accordingly is provided with a fluid inletand a filtrate outlet. The fluid process stream u, upstream of thepleated filtration tube, is introduced into the filtration capsulethrough the fluid inlet. Downstream of the pleated filtration tube, thefluid process stream d is released from the filtration capsule throughfiltrate outlet.

The materials used for the tubular housing will depend largely on itsintended application. Injection-moldable thermoplastic materials such aspolyethylene polypropylene and the like are the most likely candidates.However, the use of metals, glass, and ceramics are also contemplated.If sought for use in viral clearance of biopharmaceutical proteinproducts, the material selected should be compatible with the fluids(e.g., solvents) and environmental parameters (e.g., temperature andpressure) involved therein, and should have low protein-bindingcharacteristics. A preferred material in this regard is polypropylene.

Because filtration devices, in general, often need to satisfy severalstructural and functional criteria in the course of most filtrationprotocols, it is unlikely that its overall construction, including itshousing and any internal components, will be simple. Although a singlecontinuous and unitary structure is possible, in all likelihood thetubular housing will comprise several cooperating assembled parts whichtypically include a tubular housing that comprises an upper shell andone or two end caps.

The pleated filter tube is positioned within the tubular housing suchthat it will divide, in operation, the fluid process stream that flowsbetween the fluid inlet and the filtrate outlet. The pleated filter tubeis composed of at least one layer of the asymmetric membranes of thepresent invention. Preferably, the one or more layers are all orientedsuch that fluid introduced into said housing through the fluid inletcommences passage through each respective asymmetric membrane throughits open-side.

The pleats of the filter tube can be configured in a corrugated shape orspirally positioned and can have a loop-shaped cross section or a foldedcross-section, such as a W-shaped cross-section. As used herein, theterm “pleat” or “pleated” is intended to include all suchcross-sectional shapes. Relative to occupied volume, the pleatedstructure presents to an incoming fluid process flow more surface areathan that which would be presented by use of flat sheet. This is ofparticular advantage in consideration of the desire to maximize flux,especially when dealing with high-resolution viral clearance protocols.

The pleated filter tube is packaged within a replaceable cartridge.While it is possible, at least conceptually, to place pleated filtertube within the filter capsule without the agency of a cartridge,replaceable or otherwise, in practice, commercial and environmentaladvantages are realized by allowing the possibility of easily replacinga spent pleated filter tube, without having to undergo burdensome and/orcumbersome dismantling procedures, and/or requiring disposal of anentire filter capsule. The replacement is performed by unscrewing endcap from upper shell, unplugging a spent filter cartridge from thefiltrate outlet to which it is frictionally mated, plugging therein afresh cartridge, and screwing the cap back on.

The one or more tubular pleated sheets are maintained in a relativelyfixed tubular conformation within the filter capsule by use of theexternal and internal supports that together form the replaceablecartridge. These supports are made of rigid material and provided withuniformly dispersed holes to allow the inward flow i of fluid fromregions peripheral to the pleated filter tube, through the membranesthereof into tube's core, and then ultimately out of filter capsule.

For further details regarding the construction and functions of areplaceable filter cartridge, reference can be made to U.S. Pat. No.5,736,044, issued to S. Proulx et al. on Apr. 7, 1998. Among othersubject matter, the patent describes a composite filter cartridge thatincludes both sheet membranes and depth filters. Aspects of suchcomposite filter can be imported into the construction of the presentfilter capsule, without departing from the spirit and scope of theinvention as defined herein.

To remove virus from a protein solution, a solution containingprotein(s) of interest and one or more types of viruses and subjectingthe solution to a filtration step utilizing one or more ultrafiltrationmembranes which can be conducted either in the TFF mode or the NFF mode.In either mode, the filtration is conducted under conditions to retainthe virus generally having a 20 to 100 nanometer (nm) diameter on themembrane while permitting passage of protein(s) through the membrane. Inaddition, when filtration of the solution is completed, the membrane isflushed with water or an aqueous buffer solution to remove any retainedproteins. The use of the flushing step permits obtaining high yield ofprotein solution substantially free of virus.

EXAMPLES

Cloud Point

The visual cloudpoint temperature is used to approximate the lowercritical solution temperature for a polymeric solution of a givencomposition. This is the temperature at which a polymeric solution phaseseparates from one phase into two phases upon heating.

The procedure involves heating a small lacquer sample enclosed in atransparent container in a heating bath and observing the temperature atwhich the solution begins to turn cloudy. The procedure is performedslowly enough to ensure that the temperature indicated by a thermometerin the bath is the same as that in the lacquer sample.

Auto Ramp Bubble Point

The ABP Tester is an automated pressure-ramping device used formeasuring bubble points on ultrafiltration and microporous membranes.The ABP bubble point is the “foam-all-over” pressure, visually observedby the operator.

Vmax

Vmax is a measure of the amount of solution membrane can filter beforebeing plugged so that the flux is reduced to approximately zero flow.Vmax is measured by filtering a solution at a predetermined pressure andrecording the volume filtered as a function of time. Time divided byvolume is plotted versus volume. The inverse of the slope is Vmax.

Viral Particle Retention Testing

Testing was carried out using a single 47 mm disk in a stainless steelholder (Millipore, Billerica, Mass.) cat # XX44 047 00) at a constantpressure of 30 psid, and data was collected automatically through acomputer data acquisition package. Membranes were wet out with Milli-ROwater (Millipore Corporation, Billerica, Mass.). All trials began with abuffer flush for 2-5 minutes to equilibrate the membrane and determinepermeability. Membranes were run with their open pore side to the feedpressure. All candidates were tested with a solution containing 1 mg/mLhuman plasma IgG (Bayer, lot # 648U035) and 10⁷ pfu/mL Phi-X174(Promega, cat # I1041 m lot # 7731801) in 10 mM acetate buffer, pH 5.0.Challenge particles of bacterial phage, Phi X 174 were assayed by aplaque assay using their host bacteria. A dilution series was generatedto determine concentration. LRV was calculated as the negative logarithmof the ratio of permeate concentration to feed concentration.

Example 1

In the examples, solution preparation was done as follows.

Polyethersulfone (PES) membranes were cast from a solution consisting ofthe polymer, Polyethersulfone (PES), Radel A200 resin (Solvay) solvent,N-methylpyrrolidone (NMP) and non solvent, triethylene glycol (TEG). Thesolution was homogeneous at room temperature but phase separates whenheated. The temperature at which the solution starts to phase separatecalled the cloud point temperature and was a function of the compositionof the solution and extremely sensitive to the concentration of water.It is important to minimize the exposure of the raw materials,especially TEG, and the final solution to the atmosphere. The polymerwas predried at for example 150° C. for three hours.

The mix was made in 2 steps. First, the polymer was added to a mixtureof all the NMP and only part of the TEG. This portion was mixed whileheated to between about 50° C. and 80° C. until the solution is clear.The temperature was lowered to between about 30° C. and about 50° C. Theremaining TEG was then added to form the final solution.

A first polymer solution was prepared by dissolving 17% PES (Radel A200)in 29.2% NMP and 53.8% TEG. The resulting cloud point was 50.2° C.

A second polymer solution was prepared by dissolving 22% PES (RadelA200) in 28.1% NMP and 49.9% TEG. Additional NMP was added (6.3% offinal solution) to arrive at a cloud point of 83.6° C.

The two solutions were co-cast as described in WO 01/89673 (Kools),using a slot die coater. The cast thickness of the first solution wasadjusted to give a final layer thickness of 145 μm. The cast thicknessof the second solution was adjusted to give a final layer thickness of15 μm or about 10% of the overall membrane thickness.

The formation conditions are selected so that the first solution wasquickly heated on the casting drum, a temperature above its cloud point,before the point of immersion into an aqueous immersion bath at 55° C.At the same time the second solution does not reach a temperature of itscloud point. As a result, a formation of a microporous layer resultedfrom the first polymer solution and a formation of an ultrafiltrationlayer resulted from the second polymer solution. The final membranecharacteristics were varied by adjusting the drum temperature, and thethickness of the second layer was minimized in order to avoidundesirable macrovoid formation.

The resulting structures and properties are shown below. The retentivenature is indicated by the high bubble point, while the void-free UFlayer and dense UF surface can be clearly seen in the scanning electronmicroscope images (FIG. 4 a. and FIG. 4 b) (Drum temp 45° C. is shown).The transition from UF to MP is less clear, which could be advantageousfor maximizing throughput.

Drum Bubble Phi-X 174 Sample Temp (° C.) Point (psi) Retention (LRV) 158 102 2 56 109 0.4 3 55 108 0.5 4 50 111 1.6 5 45 112 2.8

The data in the Table show increased virus retention with decreasingdrum temperature. This effect of drum temperature is unexpected becausethe retentive UF layer has a LCST much above any of the drumtemperatures used, and it is not expected that heating the UF solutionto this degree would have any effect on membrane formation. However, ascan be seen form the data, reducing the drum temperature from 58° C. to45° C. increased virus retention by more than two orders of magnitude.

Sample 5 was compared to a two layer membrane made by casting anultrafiltration layer onto a preformed microporous membrane to form acomposite membrane with two distinct layers (Millipore PPVG membrane).The results below show that for similar permeability and BAP bubblepoints, the membrane of the present invention (Sample 5) had greatlyimproved Vmax properties and better virus removal.

Flux ABP liters/sq. Phi-X Membrane Bubble point meter/hr/psi LRV VmaxSample 5 112 58 2.8 7717 PPVG 128 45.6 2.3 437

Example 2

A first polymer solution was prepared by dissolving 22% PES (Radel A200resin) in 28.1% NMP and 49.9% TEG. The resulting cloud point was 48.6°C.

A second polymer solution was prepared by dissolving 14% PES (Radel A200resin) in 29.2% NMP and 56.8% TEG. An additional 3% of NMP was added toarrive at a cloud point of 59.3° C.

The two solutions were co-cast as described in Example 1. The castthickness of the first solution was adjusted to give a final layerthickness of 140 μm. The cast thickness of the second solution wasadjusted to give a final layer thickness of 13 μm or 8% of the overallmembrane thickness.

The formation conditions were selected so that the first solution wasquickly exposed to the heated drum at 55° C., which was above its cloudpoint, before the point of immersion into an aqueous immersion bath at45° C., which was below its cloud point. As a result, a formation of amacrovoid-free UF layer resulted from the first polymer solution and aformation of a thin microporous layer resulted from the second polymersolution.

The resulting bubble point was relatively high, and it is assumed thathigher levels can be attained by additional variances in processconditions. The resulting structures are shown below, where thevoid-free UF layer and open MP surface can be clearly seen in thescanning electron microscope images (FIGS. 4 c and 4 d).

It was very surprising to the inventors that these conditions gave amembrane with no macrovoids and a very porous microporous surface. Thedrum temperature was above the LCST of the ultrafiltration layer, butbelow the LCST of the microporous layer. (Drum temperatures below theLCST of the ultrafiltration layer gave an ultrafiltration layer withvoids.) However, the surface of the membrane of Example 2 showed a veryhigh surface porosity.

Example 3

A first polymer solution was prepared by dissolving 17% PES (Radel A200)in 29.2% NMP and 53.8% TEG. The resulting cloud point was 43° C. Thiswould be the bottom or support microporous layer.

A second polymer solution was prepared by dissolving 21% PES (RadelA200) in 37% NMP and 42% TEG. The cloud point could not be measured,being above 150° C. This would be the top ultrafiltration layer.

The two solutions were co-cast as described in WO 01/89673 (Kools). Thesecond solution layered on the first solution. The cast thickness of thefirst solution was adjusted to give a final microporous membrane layerthickness of 160 μm. The cast thickness of the second solution wasapproximately 30 μm or about 20% of the overall membrane thickness.

The formation conditions were selected so that the layered solutionswere quickly heated on the casting drum, at a range of temperaturesaround its cloud point, before the point of immersion into an aqueousimmersion bath at 55° C. The second solution did not reach a temperatureof its cloud point. As a result, a formation of a microporous layerresulted from the first polymer solution and a formation of anultrafiltration layer resulted from the second polymer solution. Thefinal membrane characteristics were varied by adjusting the drumtemperature, and the thickness of the second layer was minimized inorder to avoid undesirable macrovoid formation.

The resulting structure and properties are shown below. The retentivenature is indicated by the high retention, while the void-free UF layerand dense UF surface can be clearly seen in the scanning electronmicroscope image (Drum 50° C. is shown). The transition from UF to MP ismore observable than in example 1, yet the resulting throughput wasunaffected.

Parvovirus Drum Temp (° C.) Retention (LRV) Vmax 60 2.8 4110 55 3.7 226950 5.0 1119 45 5.1 228 40 5.5 204 35 2.7 75

As in Example 1, lowering the drum temperature increased virusretention. The LRV at 35° C. does not agree with the trend seen in otherexperiments. These conditions were repeated and the membrane producedhad LRV of 5, and Vmax of ˜20.

Example 4

A first polymer solution was prepared by dissolving 18% PES (Radel A200)in 30.2% NMP and 51.8% TEG. The resulting cloud point was 56° C. Thiswould be the bottom or support microporous layer.

A second polymer solution was prepared by dissolving 23% PES (RadelA200) in 37% NMP and 42% TEG. The cloud point could not be measured,being above 150° C. This would be the top or ultrafiltration layer.

The two solutions were co-cast as described in WO 01/89673 (Kools). Thesecond solution layered on the first solution. The cast thickness of thefirst solution was adjusted to give a final microporous membrane layerthickness of 155 μm. The cast thickness of the second solution wasapproximately 10 μm or about 6% of the overall membrane thickness.

The formation conditions were selected so that the layered solutionswere quickly heated on the heated surface, at a range of temperaturesaround its cloud point, before the point of immersion into an aqueousimmersion bath at 55° C. The second solution did not reach a temperatureof its cloud point. As a result, a formation of a microporous layerresulted from the first polymer solution and a formation of anultrafiltration layer resulted from the second polymer solution. Thefinal membrane characteristics were varied by adjusting the surfacetemperature, and the thickness of the second layer was minimized inorder to avoid undesirable macrovoid formation. The resulting propertieswere displayed below, showing both relatively high retention and highVmax at the higher surface temperatures.

Parvovirus Surface Temp (° C.) Retention (LRV) Vmax 60 4.6 1774 55 4.51339 45 5.5 260

1.) An unsupported integral multilayer composite ultrafiltrationmembrane comprised of at least a first porous membrane layer having afirst side and an equivalent second side, and at least a second porousmembrane layer having an equivalent first side and a second side, thejunction of the first layer and the second layer being integral andhaving a transition zone of porosity from said equivalent first side ofsaid second layer through said equivalent second side of said firstlayer, wherein at least one of said layers is an asymmetricultrafiltration membrane. 2.) The membrane of claim 1, wherein the firstlayer is a skinned asymmetric ultrafiltration membrane layer and thesecond layer is a microporous membrane layer. 3.) The membrane of claim1, wherein the first layer is a skinned asymmetric ultrafiltrationmembrane layer and the second layer is an asymmetric ultrafiltrationmembrane layer. 4.) The membrane of claim 1, wherein the first layer isa thin microporous ultrafiltration membrane layer and the second layeris asymmetric ultrafiltration membrane layer. 5.) The membrane of claim1, wherein the first layer is a skinned asymmetric ultrafiltrationmembrane layer and the second layer is an asymmetric ultrafiltrationmembrane layer, and wherein the first ultrafiltration membrane layer hasa MWCO higher than the second ultrafiltration membrane layer. 6.) Aprocess for forming an integral multilayered composite ultrafiltrationmembrane compromising the steps of operatingly positioning a polymersolution applying apparatus having at least two dispensing outletsrelative to a moving carrier surface, and; supplying each dispensingoutlet with a different polymer solution, and; applying said solutionsonto said moving carrier surface so as to create a multiple layercoating on said carrier, and wherein; said multiple layers are dispensedwith essentially no time interval between successive layers beingapplied, and; subjecting said multiple liquid layers to a phaseseparation process so as to form a wet multilayer ultrafiltrationmembrane. 7.) A process for forming an integral multilayered compositeultrafiltration membrane compromising the steps of operatinglypositioning a polymer solution applying apparatus having at least twodispensing outlets relative to a moving carrier surface, and; supplyingeach dispensing outlet with a different polymer solution, and; applyingsaid solutions onto said moving carrier surface so as to create amultiple layer coating on said carrier, and wherein; said multiplelayers are dispensed sequentially and the previous layer is onlypartially phase separated before each successive layer is applied, and;subjecting said multiple liquid layers to a phase separation process tocomplete phase separation and to form a wet multilayer ultrafiltrationmembrane. 8.) The process of claim 6 wherein the membrane is then dried.9.) The process of claim 6 wherein the membrane is then washed. 10.) Theprocess of claim 6 wherein the membrane is then washed and dried. 11.)The process of claim 7 wherein the membrane is then dried. 12.) Theprocess of claim 7 wherein the membrane is then washed. 13.) The processof claim 7 wherein the membrane is then washed and dried. 14.) A virusremoval methodology, suitable for conducting a high-flux fluidseparation of a virus from a protein in the course of biopharmaceuticalmanufacture, the methodology comprising the steps of: (a) providing afiltration device comprising a housing having a fluid inlet and afiltrate outlet, and containing at least one multilayered compositemembrane having at least one ultrafiltration layer produced from atleast two polymer solutions membranes, wherein: (i) said compositemembranes are each substantially hydrophilic, (ii) said compositemembranes are each capable of substantially preventing the passagetherethrough of said virus and substantially permitting the passagetherethrough of said protein, (iii) said composite membranes have each atight-side and an open-side, the average surface pore size of saidtight-side being less than the average surface pore size of saidopen-side, and (iv) the foremost composite membrane is oriented suchthat fluid introduced into said housing through the fluid inletcommences passage through said foremost asymmetric membrane through itsopen-side; (b) providing a manufactured protein-containing solution,wherein the predominant solute in said solution is said protein, andwherein the solution is prone to contamination by said virus; and (c)flowing said manufactured protein-containing solution through saidfiltration device under conditions sufficient to effect substantialpassage of said protein through each of said composite membranes and outof said housing through said filtrate outlet, whereby any of said viruscontaminating said manufactured protein-containing solution, beingsubstantially prevented from passage through said asymmetric membranes,is substantially removed therefrom. 15.) The virus removal methodologyof claim 14 wherein the composite membranes form a pleated tube. 16.)The virus removal methodology of claim 14 wherein each of said compositemembranes are substantially identical in their composition and porosity,and wherein the porosity of each of said asymmetric membranes is definedto enable performance of the virus removal methodology, yielding a logreduction value (LRV) greater than 6 and a protein passage greater than98%. 17.) A filtration capsule, suitable for use in virus removalmethodologies, comprising a tubular housing though which a fluid processstream can be conducted, the housing having a fluid inlet and a filtrateoutlet, and containing a pleated tube composed of one, two or threeinterfacially-abutting multilayered composite membranes having at leastone ultrafiltration layer produced from at least two polymer solutionsmembranes, wherein the pleated tube is positioned within said processstream between said fluid inlet and said filtrate outlet, and whereineach of said composite membranes is: (a) substantially hydrophilic, (b)capable of substantially preventing the passage therethrough of viruses,(c) provided with a tight-side and an open-side, the average pore sizeof said tight-side being less than the average pore size of saidopen-side, and (d) oriented such that fluid introduced into said housingthrough the fluid inlet commences passage through each asymmetricmembrane through its open-side. 18.) The virus removal methodology ofclaim 14 wherein the filtration device employed in said methodology isthe filtration cartridge of claim
 19. 19.) The filtration capsule ofclaim 17 wherein the asymmetric membranes are each capable ofsubstantially permitting the passage therethrough of protein. 20.) Thefiltration capsule of claim 17 wherein each asymmetric membrane iscomposed of polyethersulfone.